I. Overview Of Hemostasis And Thrombosis
Hemostasis refers to the arrest of blood loss from a damaged vessel. Following injury to a blood vessel, platelets adhere to macromolecules in the vessel's subendothelial regions, and, thereafter, aggregate to form a hemostatic plug. Platelets also stimulate local activation of plasma coagulation factors, resulting in the formation of a fibrin clot that serves to reinforce the platelet aggregate. The fibrin clot is subsequently lysed and the platelet aggregate retracts, leading to recanalization of the vessel.
The pathological process of thrombosis occurs when a fibrin clot and/or a platelet aggregate occludes a blood vessel. The location of the thrombotic occurrence plays a role in the resulting pathological effects. For example, venous thrombosis may result in the tissues drained by the vein to become edematous. By comparison, arterial thrombosis may cause ischemic necrosis of the tissue supplied by that artery. [See, e.g., Goodman and Gilman's The Pharmacological Basis of Therapeutics (8th edition, L. S. Goodman, A. Gilman, and A. G. Goodman, eds.) Macmillan Publishing Co. Inc., New York, p. 1311].
Thrombosis results in extensive morbidity and mortality and is a major medical problem both in the United States and abroad. To illustrate, arterial thrombosis contributes to the pathogenesis of myocardial infarction and stroke, two of the leading causes of death in the Western world. Moreover, venous thrombosis is responsible for approximately 300,000 hospital admissions each year in the United States.
II. Anticoagulant Therapy
The most commonly used anticoagulant agents are heparin, which is administered parenterally, and coumarin and indandione anticoagulants, which are administered orally. As will be discussed in detail hereafter, these agents are associated with severe shortcomings.
Heparin, an anionic, sulfated glycosaminoglycan present in the mast cells, is available commercially in the United States as the sodium salt. Heparin sodium, produced using either porcine intestinal mucosa or bovine lung tissue, is generally administered when an immediate anticoagulant effect is desired. Full-dose heparin therapy causes, among other things, the neutralization of thrombin, which results in the prevention of the conversion of fibrinogen to fibrin.
As with coumarin therapy (described below) the major adverse effect of heparin treatment is hemorrhage. Hemorrhagic complications have been reported in about 1-20% of patients receiving the agent, and severe cases of hemorrhage require the administration of protamine sulfate to counteract heparin's effect. In order to prevent bleeding and to determine the appropriate dose to maintain therapeutic efficacy, indicators of coagulation function (e.g., activated partial thromboplastin time) must be routinely monitored. Heparin is associated with other adverse effects, including acute thrombocytopenia and hypersensitivity reactions, that limit its usefulness in certain patient populations. [See generally, AHFS Drug Information, Gerald K. McKevoy, ed., pp. 931-37 (1995)].
The coumarin anticoagulants, which include dicumarol and warfarin sodium (e.g., warfarin sodium [Coumading.RTM., Du Pont]), are synthetic 3-substituted derivatives of 4-hydroxycoumarin, while the indandione anticoagulants (e.g., anisindione) are synthetic derivatives of indan-1,3-dione. The agents affect the synthesis of factors II, VII, IX, and X in the liver through interference with vitamin K. These orally administered agents are generally used when an immediate anticoagulant effect is not necessary or subsequent to heparin therapy.
The most frequently observed untoward effect of the coumarin and indandione agents is hemorrhage; as with heparin, this is actually an extension of the agents' pharmacological effect. Indeed, minor incidents of bleeding occur in approximately 1% of patients receiving these agents per year of therapy. Though not as common, massive hemorrhage can also occur with oral anticoagulant treatment, most frequently in the gastrointestinal tract or genitourinary region. In addition to having a relatively narrow therapeutic index, coumarin is a teratogen and cannot be administered to pregnant patients.
Because of the relatively narrow therapeutic index of these agents, frequent monitoring of laboratory indices of anticoagulant response (e.g., prothrombin time) is required; such monitoring may be complicated by the fact that hemorrhage may occur when the prothrombin time is in the normal range (often due, e.g., when occult lesions are present). Moreover, many commonly used pharmaceutical agents (e.g., metronidazole, barbiturates, and oral contraceptives) may increase or decrease the patient's response to oral anticoagulant agents, especially warfarin, necessitating close monitoring of which medications are being taken and adjusting the dose of the anticoagulant agent when appropriate. [See generally, AHFS Drug Information, Gerald K. McKevoy, ed., pp. 924-29 (1995)].
III. Models For Testing Anticoagulant Agents
As set forth above, currently used anticoagulant agents are associated with severe shortcomings. These shortcomings, coupled with the obvious need for anticoagulant therapy, has spurred considerable effort in the pharmaceutical industry to develop new safe and effective agents. However, the animal models used for testing potentially safe and efficacious anticoagulant agents have extensive limitations of their own.
Presently used animal models generally require the introduction of an injury to induce artificial thrombosis. For example, one commonly used procedure to induce thrombosis requires ligature of the inferior vena cava in rats. [See M. Barbanti et al., Thrombosis and Haemostasis 69(2)147-51 (1993); Reyers et al., "Stasis Induced Venous Thrombosis," In: Standardization of Animal Models of Thrombosis. K. Breddin and R. Zimmerman (eds.) Schattauer, Stuttgart; pp. 99-108 (1983)]. Unfortunately, this and other procedures are time consuming and invasive. Moreover, the specific experimental conditions used may influence whether thrombus formation occurs.
In addition to the inherent limitations of existing models requiring introduction of an injury to induce thrombosis, currently used animal models may not be entirely representative of thrombotic states in humans. This may be due, for example, to different sensitivities to a particular agent in the currently-used animal models than in humans because the artificially-induced injuries may activate other physiological factors that influence hemostasis. Thus, an agent that may appear to be effective in current models may be ineffective when administered to humans.
Clearly, what is needed are new methods and models for evaluating potentially useful experimental anticoagulant agents wherein the methods and models do not require the introduction of an artificial injury to induce thrombosis. The new methods and models should accurately predict the effect of the anticoagulant agents in humans and should also provide insight into the basic regulatory mechanisms of blood coagulation and the pathogenesis of human thrombosis.